The goal of Project 1 of the HIVRAD is to design vaccines intended to induce NAbs, based on accumulated and emerging knowledge of structure-function relationships within the HIV-1Env complex and of how Env proteins interact with cells of the immune system, in vitro and in vivo. We propose three inter-related Specific Aims. Aim 1: To further modify the amino acid sequence of HFV-1 envelope glycoproteins. to create novel forms that improve the immunogenicity of neutralization epitopes and facilitate structural studies. We will build on the SOSIP method for making stable, cleaved gp!40 trimers, by identifying and testing additional substitutions that stabilize gp41-gp41 interactions, by introducing other sequence changes into gp!20 and/or gp41 that are intended to create or better expose NAb epitopes, and by eliminating immunosuppressive regions of the Env complex. The latter studies will be coordinated with research outlined in Aim 2, and will lead to the creation of new immunogens for testing in small animals in Aim 3, and also in the MIMIC system within Core B. We will also make modifications to Env trimers that are intended to reduce protein heterogeneity and/or flexibility, and thereby facilitateX-ray crystallography studies to be conducted by Ian Wilson (Project 2). Aim 2: To study in vitro how to overcome immunosuppressive effects of gp!20 by the use of adjuvants and TLR activators. We will study the immunosuppressive effects of the mannose moieties of gp!20 glycans in vitro, using both human and murine cell-based systems, particularly dendritic cells (DCs). The abilities of TLR activators and other adjuvant-associated molecules to overcome these adverse effects will then be tested. We will, in addition, collaborate with the groups headed by Eric Mishkin and Sunil Ahuja, to derive additional immunologic and genetic information using the VaxDesign MIMIC system (Core B). The outcome of these experiments will facilitate the construction of new immunogens in Aim 1, and the rational design of immunization regimens in Aim 3 and Core B that are intended to maximize the antibody response to Env.Aim 3: To evaluate the immunogenicity of modified Env trimers in small animals. We will design and evaluate rabbit and mouse immunization studies that will be conducted within Core B, to determine whether the various modifications to Env glycoproteins that we identified and evaluated in Aims 1 and 2, can alter the immune response to these and co-administered antigens. We will also compare the immunogenicity of Env proteins with other antigens, alone and co-administered, to determine whether Env proteins can suppress or modify immune responses, including the IgG subclass pattern, to themselves and other antigens (HTV-1 derived or unrelated). The design of some experiments will also take into account information on adjuvants and TLR activators generated in Aim 2 and by using the VaxDesign MIMIC system in Core B. In an iterative process, the immunization experiments will themselves guide the design of additional studies to be carried out by VaxDesign using the MIMIC system (seeCore B). The overall outcome of these various studies will help design additional Env sequence modifications in Aim 1 that are intended to further improve immunogenicity or eliminate the causes of immunosuppression. RELEVANCE (See instructions): Nearly 1 % of the world's population is infected with HTV,and a preventive vaccine is urgently needed. Most efficacious vaccines elicit antibodies that can neutralize the pathogen, but current-generation HIV vaccines are not effective in this regard. Obstacles include our limited understanding of the structure and immunology of HIV-1 envelope trimers. This HIVRAD represents an innovative approach to addressing these challenges in order to provide a fundamental advance in our ability to elicit HFV-neutralizingantibodies with a vaccine.